Alternative Splicing Modulates Protein Arginine Methyltransferase-Dependent Methylation of Fragile X Syndrome Mental Retardation Protein †

Biochemical Molecular Neurobiology Laboratory, Department of Molecular Biology, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314, USA.
Biochemistry (Impact Factor: 3.02). 09/2006; 45(34):10385-93. DOI: 10.1021/bi0525019
Source: PubMed


The fragile X mental retardation protein (FMRP) is an RNA binding protein that is methylated by an endogenous methyltransferase in rabbit reticulocyte lysates. We mapped the region of methylation to the C-terminal arginine-glycine-rich residues encoded by FMR1 exon 15. We additionally demonstrated that mutation of R(544) to K reduced the endogenous methylation by more than 80%, while a comparable mutant R(546)-K reduced the endogenous methylation by 20%. These mutations had no effect on the subcellular distribution of FMRP, recapitulating previous results using the methyltransferase inhibitor adenosine-2',3'-dialdehyde. Using purified recombinant protein arginine methyltransferases (PRMTs), we showed that the C-terminal domain could be methylated by PRMT1, PRMT3, and PRMT4 in vitro and that both the R(544)-K mutant and the R(546)-K mutant were refractory toward these enzymes. We also report that truncating the N-terminal 12 residues encoded by FMR1 exon 15, which occurs naturally via alternative splicing, had no effect on FMRP methylation, demonstrating conclusively that phosphorylation of serine residue 500 (S(500)), one of the 12 residues, was not required for methylation. Nevertheless, truncating 13 additional amino acids, as occurs in the smallest alternatively spliced variant of FMR1 exon 15, reduced methylation by more than 85%. This suggests that differential expression and methylation of the FMRP exon 15 variants may be an important means of regulating target mRNA translation, which is consonant with recently demonstrated functional effects mediated by inhibiting FMRP methylation in cultured cells.

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    • "A coarse-grained elastic network model [61] only takes the Cα atom for each amino acid residue into account as “nodes” in a protein network [62]. For each nucleotide, 2 or 3 representative atoms are assigned as nodes [63, 64]. "
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    Computational and Mathematical Methods in Medicine 04/2012; 2012(17):569870. DOI:10.1155/2012/569870 · 0.77 Impact Factor
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    • "The location of the methylation site required for binding of Tdrkh to MIWI has been identified (Chen et al., PNAS 2009), but it is unclear how Tudor proteins identify binding sites in other proteins, especially those with multiple Tudor domains. Although the sites of methylation for FMRP have been identified (Stetler et al. 2006) and PRMT1 has been identified as a PRMT capable of methylating the protein in cells (Blackwell et al. 2010), additional PRMTs have been shown to methylate FMRP in vitro (Dolzhanskaya et al. 2006) and might also be capable of methylating one or more sites within FMRP in vivo. Methylation of FMRP has been shown to affect RNA association (Blackwell et al. 2010), and the RGG box is required for association with the RNA-binding protein Yb1/p50 (Blackwell and Ceman 2011), suggesting that methylation may also affect protein interactions. "
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    • "First, glycine-flanked arginine residues within RGG repeat motifs serve as target sites for Type I protein arginine methyltransferases (PRMTs), and methylation of specific arginine residues can have varied effects on a protein's RNA-Binding activity, its ability to interact with other proteins and its intracellular localization [22, 23]. Second, alternative splicing, in and around RG-rich domains has been shown to modulate both nucleic acid binding [24, 25] and protein methylation [26]. "
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